Sepsis is a serious problem in surgical and trauma patients and the leading cause of death in critically ill patients. Many septic patients die of organ failure caused by microvascular dysfunction. Our long term goal is to elucidate the mechanisms by which microvascular dysfunction occurs in sepsis and can be prevented. We propose to evaluate a new theory wherein ascorbate acts on several therapeutic targets to modulate the gene expression that is the key to microvascular dysfunction. The specific hypothesis is that vitamin C prevents septic microvascular dysfunction by inhibiting NADPH oxidase and inducible nitric oxide synthase (iNOS) expression in endothelial cells. Specific Aim 1 is to identify the redox state of vitamin C that confers protection in sepsis. Vitamin C in its reduced state (ascorbate) can be reversibly oxidized to dehydroascorbic acid (DHAA). We will compare the effectiveness of ascorbate and DHAA for raising intracellular ascorbate and preventing microvascular dysfunction and mortality in polymicrobial sepsis. Mice with impaired ability to utilize ascorbate because of deficiency in the ascorbate transporter SVCT2 and wild-type controls (129S6 mice) will be subjected to the septic insult of cecal ligation and puncture (CLP) or will be sham operated. Subsequently the mice will be injected with vehicle or else with high or low doses of ascorbate or DHAA. Ascorbate concentrations in plasma and tissues, oxidative and nitrosative stress markers in plasma, functional responsiveness of arterioles, red blood cell flow in capillaries, and survival will be measured. Specific Aim 2 is to determine if vitamin C acts on NADPH oxidase and iNOS to prevent microvascular dysfunction in sepsis. Mice that are deficient in the NADPH oxidase subunit p47phox, deficient in iNOS, or are wild-type controls (C57BL/6 mice), will be compared. The mice will undergo CLP or sham operation, be injected with vitamin C or vehicle, and then expression of sepsis-associated genes in arterioles and endothelial cells, plasma coagulation, arteriolar responsiveness and capillary perfusion will be assessed. Specific Aim 3 is to elucidate the mechanisms by which vitamin C affects endothelial permeability, NADPH oxidase, iNOS and tissue factor in microvascular endothelial cells exposed to septic insult. The effects of septic insult, ascorbate and DHAA on intracellular ascorbate concentration and endothelial cell viability and permeability will be determined. Additionally, the signaling pathways through which vitamin C regulates NADPH oxidase, iNOS and tissue factor will be elucidated by comparing the responses of cells that are replete or deficient in p47phox, Nox catalytic subunits and iNOS. Accomplishing the specific aims will define the protective role of vitamin C during sepsis and provide the basis for its use as an adjunct therapy in septic patients.Project Narrative The significance of our research is that it addresses an important problem of direct clinical relevance. This research is innovative because it will determine the effects on sepsis-associated microvascular dysfunction and mortality of vitamin C injection. Our research is also original because it will elucidate in clinically relevant models of sepsis the intracellular signaling pathways by which vitamin C regulates endothelial cell gene expression. [unreadable] [unreadable] [unreadable]